Fig 1.
Triplin acts through ethylene signaling pathway to cause a triple response phenotype in Arabidopsis seedlings.
(A) Phenotypes of 3-day-old, dark-grown Col-0 seedlings treated with 100 μM triplin, 50 μM ACC, or 1% (v/v) DMSO as a control. Scale bar represents 1 mm. (B) The chemical structure of triplin. (C) The hypocotyl length of 3-day-old, dark-grown Col-0, etr1-1, etr1-2, ein2-5 and ein3 eil1 seedlings treated with 100 μM triplin, 50 μM ACC, or 1% (v/v) DMSO as a control. Each experiment was repeated three times, more than 30 seedlings were used every time. Error bars represent SEM. ***P < 0.0001 (two-tailed Student’s t-test) indicated a significant difference of the hypocotyl length of the mutants compared to Col-0 treated with 100 μM triplin. (D) qRT-PCR analysis the expression of the ethylene response gene ERF1 treated by 1% (v/v) DMSO, 100 μM triplin, or 50 μM ACC. Each experiment was repeated three times, and error bars represent SEM.
Fig 2.
ran1-1 and ran1-2 are hypersensitive to triplin and copper can partially reverse the effects of triplin.
(A) Phenotypes of 3-day-old, dark-grown seedlings of Col-0, ran1-1 and ran1-2 treated with 10 μM triplin. (B) Triplin dose responses of Col-0, ran1-1, ran1-2 and two 35S:RAN1-GFP (ran1-2) transgenic lines. Data is the average hypocotyl length under each condition. Each experiment was repeated three times, more than 30 seedlings were used every time. Error bars represent SEM. (C) Phenotypes of 3-day-old, dark-grown seedlings of ran1-2, ran1-2 etr1-1 and ran1-2 ein2-5 treated with 100 μM triplin. (D) The phenotypes of 3-day-old, dark-grown seedlings of Col-0 treated with 100 μM triplin in the presence of 20 μM ZnSO4, 20 μM CuSO4 or H2O as a control. (E) The hypocotyl length of seedlings as described in (D). Each experiment was repeated three times, more than 30 seedlings were used every time. Error bars represent SEM. **p < 0.01 indicated the difference of the hypocotyl length between the seedlings treated with CuSO4 compared with H2O and ZnSO4 in the presence of 100 μM triplin. Scale bars represent 1 mm.
Fig 3.
Triplin can chelate copper ions in vitro.
(A) The phenotypes of 3-day-old, dark-grown seedlings of Col-0 treated with 50 μM CuSO4 in the presence of 1% (v/v) DMSO or 100 μM triplin. Scale bar represents 1 mm. (B) The root length of the seedlings as described in (A). Each experiment was repeated three times, more than 30 seedlings were used every time. Error bars represent SEM. (C) The relative copper contents of 3-day-old, dark-grown Col-0 seedlings on 0.5xMS growth medium with or without 100 μM triplin and/or 20 μM CuSO4. D.W represents dry weight of the seedlings. Each experiment was repeated three times (Error bars represent SEM). (D) MALDI-TOF-MS analysis of the mixture of equal volume of 100 μM CuSO4 and 100 μM triplin. Mr of triplin is 446.0g/mol. Mr of triplin+Cu is 510.0g/mol. *P < 0.05 and ***P < 0.0001 (two-tailed Student’s t-test) indicate a significant difference between groups of different treatments.
Fig 4.
Triplin can affect the transport of copper ions in vivo.
(A) The phenotypes of 3-day-old, dark-grown seedlings of Col-0 on different growth medium with (+) or without (-) 20 μM triplin. -Cu represents the growth medium made of all essential elements needed for plant growth except copper ion. BCS represents the growth medium made of 0.5xMS salt with 500 μM of the copper ion chelator BCS. Scale bar represents 1 mm. (B) The hypocotyl length of 3-day-old seedlings as described in (A). The difference of the hypocotyl lengths represent either the seedlings grown on -Cu or BCS medium compared to the ones grown on 0.5xMS. (C) The hypocotyl length of 3-day-old seedlings of Col-0 and 35S: RAN1-GFP lines grown in dark under different dose of triplin. (D) Triplin’s effects on ethylene-binding to ETR1 expressed in yeast. Saturable ethylene binding to intact yeast cells expressing the ethylene binding domain of ETR1 and membranes isolated from these yeast cells was measured. Ethylene binding is indicated as counts per minute (CPM). Each experiment was repeated three times, and error bars represent SEM. In (B) and (C), experiments were repeated three times, more than 30 seedlings were used every time. Error bars represent SEM. *P < 0.05, **P < 0.01, ***P < 0.0001 (two-tailed Student’s t-test) indicate a significant difference between groups of different treatments.
Fig 5.
The copper chaperone ATX1 interacts with RAN1.
(A) atx1-1 and atx1-2 are hypersensitive to triplin. The phenotypes of 3-day-old, dark-grown Col-0, atx1-1 and atx1-2 seedlings treated with 20 μM triplin are shown. Scale bar represents 1 mm. (B) Hypocotyl lengths of 3-day-old, dark-grown seedlings of Col-0, atx1-1, atx1-2, ran1-2, atx1-1 ran1-2 and two 35S:ATX1-GFP (atx1-1) transgenic lines treated with different doses of triplin are shown. Each experiment was repeated three times, more than 30 seedlings were used every time. Error bars represent SEM. (C) Subcellular co-localization of ATX1 and RAN1. ATX1-RFP and RAN1-GFP were transiently expressed in N. benthamiana leaves and observed and imaged under a confocal microscope. Scale bars represent 20 μM. (D) ATX1 interacted with RAN1 in yeast two-hybrid assay. ATX1 was fused to a GAL4 DNA-binding domain (BD) and RAN1-N (289 amino-terminal amino acids of RAN1) was ligated to a GAL4 activation domain (AD). The protein interactions were examined on cells grown on synthetic dropout (-Leu/-Trp/-His/-Ade) medium plus X-α-Gal (50mg/L) plates for 3 days. (E) Bimolecular fluorescence complementation assays showed interaction between ATX1 and RAN1 using the split luciferase system. Nicotiana benthamiana leaves were infiltrated with agrobacteria containing different construct combinations harboring both the C- and N-terminal of the luciferase fused to either ATX1 and RAN1-N or just one of them (controls). (F) Co-Immunoprecipitation assays showed interaction between ATX1 and RAN1. Nicotiana benthamiana leaves were infiltrated with agrobacteria containing ATX1-GFP/FLAG and RAN1-N-FLAG/GFP or just one of them (controls). The protein extracts were immunoblotted with anti-FLAG antibody or anti-GFP antibody.